1. Field of the Invention
The invention relates to the field of post-translational modifications of protein structure. In particular, it relates to the field of assays for one such modification that is involved in the modulation of protein function.
2. Background of the Prior Art
NO donors and endogenously produced NO exert pleiotropic effects including smooth muscle relaxation, cellular proliferation, apoptosis, neurotransmitter release, neurotoxicity, and differentiation. In mediating vasorelaxation, NO stimulates cGMP formation by binding to heme at the active site of soluble guanylyl cyclase which leads to a conformational alteration that augments enzyme activity1. NO also can interact with cysteines to form nitrosothiol adducts2-4, altering the activity of proteins including H-ras5, the olfactory cyclic nucleotide-gated channel6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)7. Nitrosothiols are exceptionally labile due to their reactivity with intracellular reducing agents such as ascorbic acid and glutathione8, as well as reduced metal ions, especially Cu(I)9, with tissue half-lives of seconds to a few minutes8.
The reversible regulation of protein function by S-nitrosylation has led to suggestions that nitrosothiols function as posttranslational modifications analogous to phosphorylation or acetylation2. The bulk of the evidence for protein regulation by S-nitrosylation has relied on in vitro experiments with NO donors, which in some cases also release other reactive oxygen species, or release NO molecules that differ in electronic structure from NO formed by NOS10. Additionally, the cytoplasm contains high concentrations of glutathione and metals which can bind NO, making it unclear whether S-nitrosylation can be elicited by endogenously produced NO. Despite these difficulties, Stamler and associates have provided evidence that certain proteins, including the ryanodine receptor11, caspase-312, and albumin13, possess nitrosothiol moieties in their endogenous state. Unfortunately, the photolytic-chemiluminescence technique employed in these studies13 was complex precluding widespread application, and it was necessary to purify candidate proteins before assessing their S-nitrosylation state. Thus there is a need in the art for new techniques for measuring this regulatorily important protein modification.
In a first embodiment of the invention a method is provided for assaying nitrosylation of protein substrates. A test sample comprising at least one protein substrate is treated with an alkylthiolating agent to block free thiol groups on the protein substrate. Nitrosothiol bonds on the protein substrate are reduced to form free thiol groups. Alkylthiolating agent is removed from the test sample. Free thiol groups on the protein substrate are reacted with a detectably tagged, activated mixed disulfide, transferring the detectable tag to the protein. The detectable tag on the protein substrate is detected.
In a second embodiment a method for screening for potential drugs useful in modulating protein nitrosylation is provided. A test sample comprising at least one protein substrate is contacted with a test compound. The test sample is treated with an alkylthiolating agent to block free thiol groups on the protein substrate. Nitrosothiol bonds on the protein substrate are reduced to form free thiol groups. Alkylthiolating agent is removed from the test sample. Free thiol groups on the protein substrate are reacted with a detectably tagged, activated mixed disulfide transferring the detectable tag to the protein. The detectable tag on the protein substrate is detected. Amount of detectable tag on the protein substrate is compared to an amount in a control sample similarly treated but not contacted with the test compound. A test compound which increases or decreases the amount of the detectable tag in the test sample relative to the control sample is identified as a modulator of protein nitrosylation.
In another embodiment of the invention a kit for measuring protein nitrosylation is provided. The kit comprises an alkylthiolating reagent and a detectably tagged, activated mixed disulfide reagent. Each reagent is in a separate compartment of the kit.
These and other embodiments of the invention which will be clear to those of skill in the art upon perusing the disclosure, provide the art with convenient methods for determining protein nitrosylation as well as with new physiologically relevant targets for the identification of new drugs involved in apoptosis, neurotoxicity, neurotransmitter release, cellular proliferation, smooth muscle relaxation, and differentiation.